TY  - JOUR
A1  - Röhlen, Desiree
A1  - Pilas, Johanna
A1  - Schöning, Michael Josef
A1  - Selmer, Thorsten
T1  - Development of an amperometric biosensor platform for the combined determination of l-Malic, Fumaric, and l-Aspartic acid
JF  - Applied Biochemistry and Biotechnology
N2  - Three amperometric biosensors have been developed for the detection of L-malic acid, fumaric acid, and L -aspartic acid, all based on the combination of a malate-specific dehydrogenase (MDH, EC 1.1.1.37) and diaphorase (DIA, EC 1.8.1.4). The stepwise expansion of the malate platform with the enzymes fumarate hydratase (FH, EC 4.2.1.2) and aspartate ammonia-lyase (ASPA, EC 4.3.1.1) resulted in multi-enzyme reaction cascades and, thus, augmentation of the substrate spectrum of the sensors. Electrochemical measurements were carried out in presence of the cofactor β-nicotinamide adenine dinucleotide (NAD+) and the redox mediator hexacyanoferrate (III) (HCFIII). The amperometric detection is mediated by oxidation of hexacyanoferrate (II) (HCFII) at an applied potential of + 0.3 V vs. Ag/AgCl. For each biosensor, optimum working conditions were defined by adjustment of cofactor concentrations, buffer pH, and immobilization procedure. Under these improved conditions, amperometric responses were linear up to 3.0 mM for L-malate and fumarate, respectively, with a corresponding sensitivity of 0.7 μA mM−1 (L-malate biosensor) and 0.4 μA mM−1 (fumarate biosensor). The L-aspartate detection system displayed a linear range of 1.0–10.0 mM with a sensitivity of 0.09 μA mM−1. The sensor characteristics suggest that the developed platform provides a promising method for the detection and differentiation of the three substrates.
Y1  - 2017
U6  - http://dx.doi.org/10.1007/s12010-017-2578-1
SN  - 1559-0291
VL  - 183
SP  - 566
EP  - 581
PB  - Springer
CY  - Berlin
ER  - 
TY  - JOUR
A1  - Pilas, Johanna
A1  - Yazici, Yasemen
A1  - Selmer, Thorsten
A1  - Keusgen, Michael
A1  - Schöning, Michael Josef
T1  - Optimization of an amperometric biosensor array for simultaneous measurement of ethanol, formate, d- and l-lactate
JF  - Electrochimica Acta
N2  - The immobilization of NAD+-dependent dehydrogenases, in combination with a diaphorase, enables the facile development of multiparametric sensing devices. In this work, an amperometric biosensor array for simultaneous determination of ethanol, formate, d- and l-lactate is presented. Enzyme immobilization on platinum thin-film electrodes was realized by chemical cross-linking with glutaraldehyde. The optimization of the sensor performance was investigated with regard to enzyme loading, glutaraldehyde concentration, pH, cofactor concentration and temperature. Under optimal working conditions (potassium phosphate buffer with pH 7.5, 2.5 mmol L-1 NAD+, 2.0 mmol L-1 ferricyanide, 25 °C and 0.4% glutaraldehyde) the linear working range and sensitivity of the four sensor elements was improved. Simultaneous and cross-talk free measurements of four different metabolic parameters were performed successfully. The reliable analytical performance of the biosensor array was demonstrated by application in a clarified sample of inoculum sludge. Thereby, a promising approach for on-site monitoring of fermentation processes is provided.
KW  - Simultaneous determination
KW  - Enzymatic biosensor
KW  - Diaphorase
KW  - Dehydrogenase
Y1  - 2017
U6  - http://dx.doi.org/10.1016/j.electacta.2017.07.119
SN  - 0013-4686
VL  - 251
SP  - 256
EP  - 262
PB  - Elsevier
CY  - Amsterdam
ER  -